Project Summary West Nile virus (WNV) is an emerging, neurotropic virus of the Flavivirus genus that is transmitted to humans through the bite of an infected mosquito. Flaviviruses include globally important pathogens, such as dengue (DENV) and Zika (ZIKV) virus which infect hundreds of millions yearly. Currently, there are no specific antiviral treatments for any flavivirus. Given options that interactions utilized flaviv interacted significant functionally the that three the continuing spread of flaviviruses across the globe and the dearth of to prevent or treat them, it is imperative that we develop a better understanding the host processes impact infection. W e used an affinity purification/mass spectrometry approach to identify the physical that occur between WNV and host proteins. In collaboration with Nevan Krogan at UCSF, we the data from parallel studies of DENV and ZIKV to focus on host proteins targeted by multiple iruses. We discovered 259 high- confidence WNV -interacting host proteins; of those, 49 host proteins with the analogous viral protein in either DENV or ZIKV. This analysis revealed that the most overlap between flavivirus-host interactions was for capsid- and NS5-interacting proteins. To define shared interactors that are most important for infection, we employed an RNAi screen in context of WNV, DENV and ZIKV infection. This revealed 23 factors that impacted WNV infection , 12 impacted WNV and at least one additional flavivirus, and 8 host proteins influencing infection for all flaviviruses.Among these, we identified USP15, a host deubiquitylase that interacts with the flavivirus NS5 proteins and is implicated in the induction of Type I interferons in response to viral infection. We show that USP15 is required for infection and acts as a negative regulator of Type I interferon. We will establish the requirement for the interaction between USP15 and WNV NS5 in the phenotypes we observe by using mutagenesis to identify the residues in USP15 that are critical for the NS5 interaction and will generate NS5- binding deficient USP15 mutants to test in our studies. Given that USP15 is a deubiquitylase and that several steps in the Type I interferon response require ubiquitylation, we propose that USP15 promotes infection through deubiquitylation and inhibition of Type I interferon signaling. To test this, we will monitor activation and ubiquitylation of known targets in the interferon signaling pathway upon knockdown of USP15 and determine if the USP15-NS5 interaction affects this response. We have also identified the flavivirus capsid-interacting host protein WIBG. WIBG is an RNA-binding protein involved in nonsense-mediated RNA decay (NMD) and is antiviral in flavivirus infection. We show that NMD is inhibited in flavivirus infection; moreover, depletion of the canonical NMD factor UPF1 indicates that NMD restricts flavivirus infection. We propose that NMD is an antiviral host process that is antagonized by flaviviruses. We will determine the mechanism by which NMD inhibits WNV infection and determine how the WIBG-capsid interaction influences this process. The goal of this proposal is to uncover the mechanisms by which flaviviruses subvert host innate antiviral mechanisms.